Choose your preferred view mode

Please select whether you prefer to view the MDPI pages with a view tailored for mobile displays or to view the MDPI
pages in the normal scrollable desktop version. This selection will be stored into your cookies and used automatically
in next visits. You can also change the view style at any point from the main header when using the pages with your
mobile device.

1. Introduction

The genus Tagetes (Asteraceae) is mainly native to the central and southern part of America. It consists of approximately 30 species [1]. Members of the genus Tagetes have a long history of human use as beverages, condiments, ornamentals, and medicinal decoctions. Tagetes minuta L. has been used as anthelmintic, diuretic, antispasmodic, and for treatment of stomach and intestinal diseases [2]. Tagetes oil is used as a flavor component in food products including cola and alcoholic beverages, frozen dairy desserts, candy, baked goods, gelatins, puddings, condiments, and relishes [3]. The oil has antibacterial [4], larvicidal [5], and insecticidal [6] activities. Previous phytochemical studies of T. minuta L. led to the isolation of terpenes [2,7], flavonoids [8], thiophenes, and aromatic compounds [9]. This article reports the isolation and characterization of two new compounds: 5-methyl-2,2',5',2'',5'',2''',5''',2''''-quinquethiophene (1) and quercetagetin-6-O-(6-O-caffeoyl-β-d-gluco-pyranoside) (9), together with seven known flavonoids (Figure 1).

Figure 1.
Chemical structures of the isolated compounds 1–9.

Figure 1.
Chemical structures of the isolated compounds 1–9.

2. Results and Discussion

Compound 1 was isolated as brown needles. HRESIMS gave an [M+H]+ at m/z 427.6611 and 428.6613 [M+2H]+, which is consistent with the molecular formula C21H14S5, implying fifteen degrees of unsaturation. The UV absorption maxima at 387 and 334 nm indicated the presence of quinquethiophene moiety [10,11]. The 1H-NMR spectrum showed eleven protons signals at δH 6.64–7.21 with coupling constants 5.5–3.5 Hz characteristic for 5-subsituted quinquethiophenes [11]. Additionally, the proton signal at δH 2.42 (3H, s) indicated the presence of a methyl group (Table 1). The 13C-NMR spectrum exhibited twenty one carbon resonances. The multiplicities of the carbons in 1 were confirmed with DEPT and HSQC experiments, which showed one methyl, eleven methines, and nine quaternary carbons. 1H-1H COSY provided five spin systems for five thiophene rings (Figure 2). The HMBC spectrum exhibited cross peaks from methyl protons at C-5 to C-4 and C-5. In HMBC spectrum, cross-peaks from H-3 to C-2', H-3' to C-2, H-4' to C-2'', H-3'' to C-5', H-4'' to C-2''', H-3''' to C-5'', H-4''' to C-2'''', and H-3'''' to C-5'''confirmed the connectivity of thiophene rings [11]. Accordingly, 1 was 5-methyl-2, 2',5', 2'',5'',2''',5''',2''''-quinquethiophene. Compound 1 was isolated for the first time from natural origin.

The 13C-NMR spectrum displayed fifteen carbon signals were attributed to quercetagetin skeleton [16,17] and six carbons for glucose. The multiplicity of each carbon was determined by HSQC experiment. The glucose moiety was located at C-6 based on the HMBC cross peak of H-1'' at δH 5.02 (1H, d, J = 6.5 Hz) to C-6 (δC 129.6) and further confirmed by its reaction with diagnostic shift reagents. In the HMBC spectrum, the methylene protons at δH 4.41 (H-6''B) and 4.30 (H-6''A) correlated with the caffeoyl carbonyl group at δC 166.5 suggesting the connectivity of caffeoyl moiety at C-6'' and confirmed by the downfield shift of C-6'' (δC 64.6). Acid hydrolysis of 9 afforded quercetagetin, caffeic acid, and β-d-glucose. They were identified by co-chromatography with authentic samples using (S5) [14]. Accordingly, 9 was identified as quercetagetin-6-O-(6-O-caffeoyl-β-d-glucopyranoside).

The other compounds were identified as quercetin-3,6-dimethyl ether (2) [18], quercetin-3-methyl ether (3) [18], quercetin (4) [18], axillarin-7-O-β-d-glucopyranoside (5) [19], quercetagetin-3,7-dimethoxy-6-O-β-d-glucopyranoside (6) [20], quercetagetin-7-methoxy-6-O-β-d-glucopyranoside (7) [20], and quercetagetin-6-O-β-d-glucopyranoside (8) [20] by comparison of their physical and spectral data with those in the literature. The antioxidant activity of the isolated compounds 2-9 was determined by using a DPPH free radical scavenging system. The antioxidant percentage activity ranged from 91.6 to 68.3% (Table 3). The antioxidant effect of these compounds was related to the number of free phenolic hydroxyl groups in the 3,4-dihydroxy form in their structures, which explains the close similarity of their antioxidant activity. Absence or blocking of the hydroxyl groups by a methyl or glucose moiety leads to a decrease of the antioxidant activity [21].

3.2. Plant Material

The leaves of Tagetes minuta L. (Asteraceae) were collected in June 2012 from Al-Baha, Saudi Arabia. The plant was identified by Dr. A. A. Fayed, Prof. of Plant Taxonomy, Faculty of Science, Assiut University, Egypt. A voucher specimen (TM-1-2012) was deposited at the herbarium of the research center for medicinal, aromatic and poisonous plants, King Saud University.

3.5. Acid Hydrolysis of 9

Compound 9 (3 mg) was refluxed in 10 mL of 1 N HCl for 4 h. The aglycone was extracted with CHCl3. The sugar in the aqueous layer was identified by co-paper chromatography (PC) with authentic materials using solvent system (S5) and aniline phthalate spray as detection reagent [14].

3.7. Antimalarial Assay

The isolated compounds were tested on chloroquine sensitive (D6, Sierraleon) and resistant (W2, Indo-china) strains of Plasmodium falciparum using previously reported method [22,25]. Artemisinin and chloroquine were included in each assay as anti-malarial drug controls.

3.8. Antileishmanial Assay

The anti-leishmanial activity of the isolated metabolites was tested in vitro against a culture of L. donovani promastigotes as previously outlined [26]. Pentamidine and amphoterecin B were used as positive standards.

4. Conclusions

In conclusion, in this study nine compounds were isolated and elucidated from T. minuta L. two of them (compounds 1 and 9) are new. The antioxidant, antimicrobial, antimalarial, and antileishmanial activities of the isolated compounds were evaluated. They showed antioxidant activity ranging from 91.6% to 68.3%. Compound 8 showed weak antileishmanial activity with an IC50 31.0 μg/mL, while compound 3 showed moderate antimalarial activity against chloroquine sensitive (D6) clones of P. falciparum with an IC50 4.37 μg/mL.

Acknowledgments

The authors are grateful to the research center for female scientific and medical colleges, deanship of scientific research in King Saud University for the financial support and to Volker Brecht (Nuclear Magnetics Resonance, Institute fuer Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Germany) for HRMS measurements. Also, the authors are thankful to Melissa Jacobs, Shabana Khan, and Babu Tekwani for antimicrobial, anti-malarial, and anti-leishmanial testing.